The anaerobic digestion of mixed indigenous microalgae, grown in a secondary effluent, was evaluated in batch tests at mesophilic (35°C) and thermophilic (50°C) conditions. Under mesophilic conditions, specific methane production varied from 178 to 207 mL CH4/g volatile solids (VS) and the maximum production rate varied from 8.8 to 26.1 mL CH4/(gVS day), depending on the type of microalgae culture. Lower methane parameters were observed in those cultures where Scenedesmus represents more than 95% of the microalge. The culture with the lowest digestion performances under mesophilic conditions was studied under thermophilic conditions. The increase in the incubation temperature significantly increased the specific methane production (390 mL CH4/g VS) and rate (26.0 mL CH4/(gVS day)). However, under thermophilic conditions a lag period of 30 days was observed.

INTRODUCTION

The interest in microalgae-based biofuels has spread in recent years due to the increase in fossil fuel prices and the adverse effects of global climate change (Chisti 2013). Nevertheless, microalgae-based biodiesel requires an energy input derived from fossil fuels for biomass production, turning the microalgae-based biofuels into an expensive and high-energy process. Contrasting to biodiesel generation, biomethane seems to be an attractive alternative due to the direct use of the collected biomass, avoiding drying, which is the most energy-consuming step in biodiesel production (Chisti 2013). However, a life-cycle assessment showed that the production of methane from microalgae strongly correlates with the electric consumption and suggests the need of improvements for a more efficient anaerobic digestion process (Collet et al. 2011). Also, other authors identify the biomass productivity and the cost of the culture system as the major difficulties found for a sustainable methane production (Zamalloa et al. 2011; Chisti 2013). A potential alternative to decrease the costs associated with microalgae cultivation is by coupling the wastewater (WW) treatment to the algal cultivation. The growth of axenic microalgae cultures has been reported using WW (Abdel-Raouf et al. 2012; Caporgno et al. 2015) and secondary effluents (Passos et al. 2015; Yu et al. 2015). Secondary effluents are a good alternative to microalgae growth because of the content of inorganic nitrogen and phosphorus still present. These compounds may cause eutrophication if they are released to the environment. The microalgae culture offers then an interesting step for WW polishing coupled to the production of potentially valuable biomass (Abdel-Raouf et al. 2012; Caporgno et al. 2015).

Information about the anaerobic digestion of microalgae under mesophilic (35–36 °C) (Ras et al. 2011; Passos et al. 2015) or thermophilic conditions (55 °C) exists (Zamalloa et al. 2012). The majority of the studies have been conducted using axenic or non-axenic microalgae cultures (Ras et al. 2011; Alzate et al. 2012; Zamalloa et al. 2012; Markou et al. 2013). However, few data are available concerning the digestibility and methane production of mixed microalgae grown on secondary effluents. The aim of this study was to assess the methane production from the anaerobic digestion of a mixed culture of microalgae grown in a secondary effluent from a municipal wastewater treatment plant (WWTP). Four mixed microalgae were evaluated under mesophilic conditions and one of these cultures was evaluated under thermophilic conditions.

METHODS

Culture of mixed microalgae

Mixed microalgae were collected from four different aquatic environments at Querétaro, México, according to Cea-Barcia et al. (2014). To promote growth of freshwater microalgae the samples (denoted here as A, B, C and D) were initially grown in Bold's medium, which is a broad spectrum medium for freshwater microalgae of the classes Chlorophyceae, Xanthophyceae, Chrysophyceae and Cyanophyceae (Barsanti & Gualtieri 2006). After the propagation step, mixed microalgae were grown using a secondary effluent from a WWTP. Samples for characterization were taken at the end of the propagation period (3 months). An optical microscope (Leica DM500, Japan) with the Leica LAS EZ 2.0.0 software was used for microalgal identification. Genera of the four microalgae cultures were identified according to Wehr & Sheath (2003). The cell number was determined by direct counting with a 0.1 mm improved Neubauer counting chamber.

Mesophilic and thermophilic anaerobic inoculum

The mesophilic anaerobic inoculum was collected from an anaerobic reactor operated at 35 °C, and treating brewery WW. The solids content was 27 g total solids (TS)/L and 19 g volatile solids (VS)/L. The specific methanogenic activity was 17 mL CH4/(gVS day), utilizing 1.0 g chemical oxygen demand (COD)/L of glucose with 6.5 g VS/L of inoculum. The thermophilic anaerobic inoculum was collected from a thermophilic anaerobic reactor digesting activated sludge and operating between 50 and 53 °C. The solids content was 18 g TS/L and 11 g VS/L. The specific methanogenic activity was 7 mL CH4/(gVS day), utilizing 1.0 g COD/L of glucose with 6.5 g VS/L of inoculum.

The thermophilic inoculum was first maintained under endogenous anaerobic conditions and mixing for 14 days to reduce the original substrate. Next, the inoculum was fed with cellulose (3 g/L) for 6 months at 53 °C. The experiment was carried out in a batch reactor, filled with 1.5 L of thermophilic inoculum. The cellulose degradation was follow by measuring the soluble COD in the reactor. When 80% of the initial COD was consumed, the reactor was fed again with cellulose. The duration of each batch cycle lasted from 30 to 35 days. This procedure was conducted for 6 months. Finally, and before the evaluation of the methane production assays with microalgae, the optimal thermophilic temperature was determined with the adapted sludge. The specific methane production rate of the cellulose-enriched sludge was measured using glucose as substrate (3 g COD/L) and 45, 50 and 53 °C. The tests were performed in serum bottles of 120 mL with a liquid volume of 60 mL and 6.4 ± 1.2 g VS/L.

Mixed microalgae anaerobic digestion batch tests

The anaerobic digestion batch tests were performed in duplicate to determine both the microalgae biodegradation and the specific methane production rate. At mesophilic (35 °C) conditions mixed microalgae A, B, C and D were tested. For the thermophilic condition (50 °C) only mixed microalgae A was tested. The experiments were carried out for 30 days at mesophilic conditions and 55 days at thermophilic conditions. The tests were performed in serum bottles of 120 mL filled with 60 mL of a mixture of anaerobic inoculum and microalgae at a inoculum to substrate ratio of 0.5 (gVSinoculum/gVSmicroalgae), as suggested by Alzate et al. (2012), and with solids content of 12 ± 2 g TS/L. The bottles were gassed with nitrogen for 1 min, closed with butyl septa, sealed with aluminum caps and incubated either at mesophilic (35 °C) or at thermophilic conditions (50 °C) using orbital mixing at 100 rpm. The biogas production was monitored by periodic measurements of the pressure at the headspace and biogas composition was determined by chromatography. Blank tests containing 60 mL of inoculum were carried out to determine the endogenous methane production from the inoculum. The endogenous methane production was subtracted from the total methane production. The methane volume produced was expressed at 273 K and 1 atm. Microalgae biodegradation was calculated as the ratio of the experimental to the theoretical methane production, the latter estimated assuming a theoretical production of 350 mL CH4/g COD degraded. The experimental value was evaluated considered the maximum methane production.

A kinetic analysis of cumulative methane production was performed using the modified Gompertz equation (Equation (1)). This equation has been widely used to model gas production data (Barakat et al. 2014). 
formula
1
where Mmax is the maximum methane production (mL/g VS), Rmax is the maximum methane production rate (mL/(gVS day)), λ is the lag-phase time before the exponential methane production (day) and t is the incubation time (day).

Analytical methods

TS, VS and COD concentrations were determined according to Standard Methods (APHA 2005). The pressure in the headspace of the serum bottles was measured with a pressure transducer (Model PS 100-2bar). Carbon dioxide and methane were analyzed with a gas chromatograph (SRI 8610C) equipped with a thermal conductivity detector and two packed columns (6 ft × 1/8 in, silica gel packed column and 6 ft × 1/8 in, molecular sieve 13 × packed column). The injector and detector temperatures were 90 °C and 150 °C, respectively. The initial column temperature was 40 °C, which was held for 4 min and then gradually increased to 110 °C at a rate of 20 °C/min. The final column temperature was held for 3 min. Nitrogen was used as a carrier gas at a flow rate of 20 mL/min. Liquid samples were taken at the end of the experiments for the analysis of volatile fatty acids (VFAs). One millilitre of sample was centrifuged at 600 g for 5 min. VFAs concentrations were determined using a chromatograph (Varian 3300) fitted with a flame ionization detector and a 15 m long (0.53 mm id) Zebron ZB-FFAP column. Injector and detector temperatures were maintained at 190 °C and 210 °C, respectively. The temperature of the column was maintained at 45 °C for 1.5 min; then, it was increased to 135 °C at a rate of 8 °C/min. The carrier gas was nitrogen at 9.5 mL/min.

Statistical analysis

Statistical significance was determined by analysis of variance (P = 0.05) using Matlab (Version 2011 B). Experimental data were presented as average values ± standard deviations (error bars in figures).

RESULTS AND DISCUSSION

Culture of mixed microalgae

The mixed microalgae (A) was composed of Scenedesmus (98%), Keratococcus (1%) and undetermined (<1%); mixed microalgae (B) was composed of Scenedesmus (95%), Keratococcus (4%), Closterium and undetermined (<1%); mixed microalgae (C) was composed of Ulothrix (50%), Oscillatoria (30%), Scenedesmus (19%), Anabaena, Oocystis and undetermined (<1%); and mixed microalgae (D) was composed of Scenedesmus (21%), Keratococcus (29%), Golenkinia (42%), Monoraphidium (7%), Oscillatoria and undetermined (<1%) (Figure 1).

Figure 1

Mixed microalgae A (a), B (b), C (c) and D (d) obtained after 3 months of cultivation in secondary effluent of a WWTP.

Figure 1

Mixed microalgae A (a), B (b), C (c) and D (d) obtained after 3 months of cultivation in secondary effluent of a WWTP.

Mesophilic assays

The microalgae biodegradation and methane production (Mmax and Rmax) were determined at 35 °C for the mixed microalgae A, B, C and D. No accumulation of VFAs was found in all the experiments. Figure 2 shows the specific methane production kinetics under mesophilic conditions, and Table 1 shows the anaerobic digestion parameters. The maximum methane accumulated and the lag time were similar for all the consortia (178–207 mL CH4/g VSmicroalgae and lag times <1 h). However, differences were found for the Rmax values. Culture D presented the highest Rmax (26.1 ± 2.3 mL CH4/(gVS day)), followed by culture C (14.8 ± 0.4 mL CH4/(gVS day)). Lower Rmax was obtained for cultures A and B (8.8 ± 0.7 mL CH4/(gVS day) and 9.9 ± 0.7 mL CH4/(gVS day), respectively). For the case of cultures A and B, Scenedesmus represented more than 95% of the microalgae in the cultures. The lower rates can be explained considering that Scenedesmus possesses a rigid cell wall (Takeda 1996) with a high resistance to disruption (Blumreisinger et al. 1983). On the other hand, it has been observed that Scenedesmus can grow at 35 °C (Hodaifa et al. 2010). That may hinder the hydrolysis process under mesophilic conditions (Zamalloa et al. 2012). Also, Monoraphidium (Chlorophyceae) was present in cultures A and B, characterized by a lower rigidness in its cell wall structure (Blumreisinger et al. 1983). In contrast, cultures C and D are constituted of microalgae belonging to taxa Ulvophyceae (Ulothrix) and Trebouxiophyceae (Keratococcus). These microalgae do not possess rigid cell walls or pectin–cellulose complexes in their structure, thus facilitating the biodegradation (Domozych et al. 2012).

Table 1

Anaerobic digestion parameters of four mixed microalgae at 35 °C. Standard deviation corresponds to duplicate batch tests

Microalgae 
Rmax, mL CH4/(gVS d) 8.8 ± 0.7 9.9 ± 0.7 14.8 ± 0.4 26.1 ± 2.3 
Mmax, mL CH4/gVSmicroalgae 178 ± 10 204 ± 7 196 ± 0.8 207 ± 16 
λ, day <1 <1 <1 <1 
CH4 composition, % 62 ± 0.6 62 ± 0.1 64 ± 0.4 66 ± 0.2 
Biodegradation, % 27 ± 1 35 ± 1 35 ± 0.1 31 ± 2 
Microalgae 
Rmax, mL CH4/(gVS d) 8.8 ± 0.7 9.9 ± 0.7 14.8 ± 0.4 26.1 ± 2.3 
Mmax, mL CH4/gVSmicroalgae 178 ± 10 204 ± 7 196 ± 0.8 207 ± 16 
λ, day <1 <1 <1 <1 
CH4 composition, % 62 ± 0.6 62 ± 0.1 64 ± 0.4 66 ± 0.2 
Biodegradation, % 27 ± 1 35 ± 1 35 ± 0.1 31 ± 2 
Figure 2

Methane production kinetics of mixed microalgae A, B, C and D at 35 °C. Lines represent the fitting of the Gompertz model. Standard deviation corresponds to duplicate batch tests.

Figure 2

Methane production kinetics of mixed microalgae A, B, C and D at 35 °C. Lines represent the fitting of the Gompertz model. Standard deviation corresponds to duplicate batch tests.

The microalgal biodegradation was 27%, 35%, 35% and 31% for cultures A, B, C and D, respectively, and can be explained by the cell wall resistance. It has been found that the application of a pretreatment can enhance the break-up of the cell walls of microalgae. For example, thermal hydrolysis (Alzate et al. 2012) increased the microalgal biodegradation up to 60% and hydrothermal liquefaction (Tommasso et al. 2015) up to 84%.

Determination of thermophilic reaction temperature

Thermophilic conditions were applied to enhance the anaerobic digestion of mixed microalgae. As the microalgae possess a cell wall composed mainly of cellulose, it was decided to adapt the thermophilic sludge to cellulose for 6 months. The activity of the cellulose-enriched sludge was evaluated after the adaptation period. For that, glucose was used as substrate, and three temperatures were tested (45, 50 and 53 °C). It was found that the adaptation phase increased the sludge activity. The specific methane production, using glucose as substrate, changed from 7 mL CH4/(gVS day) (for the original sludge) to 14.7 ± 1.3, 20.0 ± 0.5 and 15.5 ± 0.5 mL CH4/(gVS day) when 45 °C, 50 °C and 53 °C were evaluated after adaptation, respectively.

Figure 3 shows the methane production kinetics for the tested temperatures. The highest values for the Gompertz parameters were obtained for 50 and 53 °C (Table 2). It was found that the value of Rmax obtained at 50 °C was significantly higher than the value obtained at 53 °C (P = 0.01; P < 0.05). For this reason, and considering that at 50 °C less energy is required than at 53 °C, the former was chosen as the thermophilic condition to be tested.

Table 2

Gompertz parameters obtained with the cellulose-enriched sludge and glucose as substrate for three different temperatures

Temperature 45 °C 50 °C 53 °C 
Rmax, mL CH4/(gVS day) 5.6 ± 0.5 7.6 ± 0.2 5.9 ± 0.2 
Mmax, mL CH4/g VS 45 ± 0.9 52 ± 2.0 58 ± 0.7 
λ, day 0.6 1.9 1.3 
Temperature 45 °C 50 °C 53 °C 
Rmax, mL CH4/(gVS day) 5.6 ± 0.5 7.6 ± 0.2 5.9 ± 0.2 
Mmax, mL CH4/g VS 45 ± 0.9 52 ± 2.0 58 ± 0.7 
λ, day 0.6 1.9 1.3 
Table 3

Anaerobic digestion parameters of mixed microalgae A at 50 °C. Standard deviation corresponds to duplicate batch tests

 50 °C (0–30 day) 50 °C (>30 day) 
Rmax, (mL CH4/(gVS day)) 4.0 ± 0.4 26 ± 3 
Mmax, (mL CH4/gVS) 55 ± 6 390 ± 22 
λ, (day) 30 
CH4 composition (%) 52 ± 0.9 69 ± 0.8 
Biodegradation (%) – 56 ± 3 
 50 °C (0–30 day) 50 °C (>30 day) 
Rmax, (mL CH4/(gVS day)) 4.0 ± 0.4 26 ± 3 
Mmax, (mL CH4/gVS) 55 ± 6 390 ± 22 
λ, (day) 30 
CH4 composition (%) 52 ± 0.9 69 ± 0.8 
Biodegradation (%) – 56 ± 3 
Figure 3

Methane production kinetics for glucose under thermophilic conditions at 45, 50 and 53 °C. Lines represent the fitting of the Gompertz model. Standard deviation corresponds to duplicate batch tests.

Figure 3

Methane production kinetics for glucose under thermophilic conditions at 45, 50 and 53 °C. Lines represent the fitting of the Gompertz model. Standard deviation corresponds to duplicate batch tests.

Thermophilic assays

Culture A, mainly composed of Scenedesmus, was selected for thermophilic tests because of the low methane production rate and biodegradation observed under mesophilic conditions. In addition, culture A presented the highest biomass productivities when this culture was grown in secondary effluents (Cea-Barcia et al. 2014).

A biphasic behavior was observed under thermophilic digestion of microalgae A, for the two replicas (Figure 4). Low methane production was observed during the first 30 days, giving an Rmax of 4.0 mL CH4/(gVS day), and representing a half of that observed under mesophilic conditions. However, after 30 days, methane production increased substantially, with an Rmax value of 26 mL CH4/(gVS day). That value is three times higher than the Rmax observed under mesophilic conditions. Similarly, Mmax and biodegradation were increased by a factor of 2.1 and 2.2, respectively (Table 3). The biphasic behavior is explained by considering that enzymatic hydrolysis may occur during the first 30 days of the thermal treatment. Enzymatic hydrolysis has been suggested (Carrère et al. 2010) to be the main mechanism occurring in thermal treatments at moderate temperatures (<70 °C). Thermal pretreatments improved the methane production rate and yield because of solubilization of the microalgal biomass. Also, the degree of solubilization increased with the temperature and the incubation time (Passos et al. 2013).

Figure 4

Methane production kinetics of mixed microalgae A at 50 °C. Line represents the fitting of the Gompertz model.

Figure 4

Methane production kinetics of mixed microalgae A at 50 °C. Line represents the fitting of the Gompertz model.

Even though the thermophilic sludge was adapted to cellulose and presented satisfactory methane production rates with glucose, results indicated that the microalgae constituted a more complex substrate requiring long degradation periods of 30 days. However, the results evidenced an increase in the biodegradation and methane production, turning to thermophilic digestion in an attractive process. The feasibility of methane production from mixed microalgae under thermophilic conditions still needs to be optimized and studied, by evaluating the process in continuous reactors.

CONCLUSIONS

The anaerobic digestion of mixed indigenous microalgae, grown in a secondary effluent, was evaluated in batch tests at mesophilic and thermophilic conditions. Under mesophilic conditions low methane production was observed in those cultures where Scenedesmus represented more than 95% of the microalgae in the culture. The culture with the lowest digestion performances under mesophilic conditions was studied under thermophilic conditions. It was found that the increase in the incubation temperature significantly increased specific methane production and rate. However, biogas production under thermophilic conditions suffered from a lag period of 30 days. The results indicated the need for further studies to evaluate the process in a continuous reactor in order to optimize the microalgae digestibility under thermophilic conditions.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial support of the project FOMIX-CONACYT-Queretaro (grant no. 192341). Jaime Perez Trevilla is acknowledged for technical assistance and DGAPA-UNAM for the postdoctoral fellowship to G. Cea-Barcia.

REFERENCES

REFERENCES
Abdel-Raouf
N.
Al-Homaidan
A. A.
Ibraheem
I. B. M.
2012
Microalgae and wastewater treatment
.
Saudi Journal of Biological Sciences
19
(
3
),
257
275
.
Alzate
M. E.
Muñoz
R.
Rogalla
F.
Fdz-Polanco
F.
Pérez-Elvira
S. I.
2012
Biochemical methane potential of microalgae: influence of substrate to inoculum ratio, biomass concentration and pretreatment
.
Bioresource Technology
123
,
488
494
.
APHA
2005
Standard Methods for the Examination of Water and Wastewater
.
21st edn
.
American Public Health Association/American Water Works Association/Water Environment Federation
,
Washington, DC
,
USA
.
Barakat
A.
Kadimi
A.
Steyer
J. P.
Carrère
H.
2014
Impact of xylan structure and lignin–xylan association on methane production from C5-sugars
.
Biomass and Bioenergy
63
,
33
45
.
Barsanti
L.
Gualtieri
P.
2006
Algae. Anatomy, Biochemistry & Biotechnology
.
Taylor & Francis
,
Boca Raton, FL, USA
.
Blumreisinger
M.
Meindl
D.
Loos
E.
1983
Cell wall composition of chlorococcal algae
.
Phytochemestry
22
(
7
),
1603
1604
.
Caporgno
M. P.
Taleb
A.
Olkiewicz
M.
Font
J.
Pruvost
J.
Legrand
J.
Bengoa
C.
2015
Microalgae cultivation in urban wastewater: nutrient removal and biomass production for biodiesel and methane
.
Algal Research
10
,
232
239
.
Carrère
H.
Dumas
C.
Battimelli
A.
Batstone
D. J.
Delgenès
J. P.
Steyer
J. P.
Ferrer
I.
2010
Pretreatment methods to improve sludge anaerobic degradability: a review
.
Journal of Hazardous Materials
183
,
1
15
.
Chisti
Y.
2013
Constraints to commercialization of algal fuels
.
Journal of Biotechnology
167
,
201
214
.
Collet
P.
Hélias
A.
Lardon
L.
Ras
M.
Goy
R.-A.
Steyer
J.-P.
2011
Life cycle assessment of microalgae culture coupled to biogas production
.
Bioresource Technology
102
,
207
214
.
Domozych
D. S.
Ciancia
M.
Fangel
J. U.
Mikkelsen
M. D.
Ulvskov
P.
Willats
W. G. T.
2012
The cell walls of green algae: a journey through evolution and diversity
.
Frontiers in Plant Science
3
,
1
7
.
Hodaifa
G.
Martínez
M. E.
Sánchez
S.
2010
Influence of temperature on growth of Scenedesmus obliquus in diluted olive mill wastewater as culture medium
.
Engineering in Life Sciences
10
(
3
),
257
264
.
Passos
F.
Gutiérrez
R.
Brockmann
D.
Steyer
J. P.
García
J.
Ferrer
I
.
2015
Microalgae production in wastewater treatment systems, anaerobic digestion and modelling using ADM1
.
Algal Research
10
,
55
63
.
Ras
M.
Lardon
L.
Sialve
B.
Bernet
N.
Steyer
J.-P.
2011
Experimental study on a coupled process of production and anaerobic digestion of Chlorella vulgaris
.
Bioresource Technology
102
,
200
206
.
Takeda
H.
1996
Cell wall sugars of some Scenedesmus species
.
Phytochemistry
42
(
3
),
673
675
.
Wehr
J. D.
Sheath
R. G.
2003
Freshwater Algae of North America: Ecology and Classification. Academic Press
.
Amsterdam
.
Zamalloa
C.
Vulsteke
E.
Albrecht
J.
Verstraete
W.
2011
The techno-economic potential of renewable energy through the anaerobic digestion of microalgae
.
Bioresource Technology
102
(
2
),
1149
1158
.